GP5^(Δ84-119)稳定表达对猪繁殖与呼吸综合征病毒复制的影响

The Effect of GP5^(Δ84-119) Stable Expression on the Replication of Porcine Reproductive and Respiratory Syndrome Virus

为探讨猪繁殖与呼吸综合征病毒(PRRSV)主要结构蛋白GP5的第二胞外区在病毒复制中的作用及机制,利用PiggyBac Transposon System Vectors构建了表达删除84—119氨基酸残基的截短型GP5的重组质粒(pPBGP5Δ84-119),转染Marc-145细胞通过嘌呤霉素抗性筛选和3次亚克隆,筛选稳定表达GP5Δ84-119的Marc-145细胞系,并利用cck-8试剂盒检测细胞的增殖情况;用PRRSV SD16株感染筛选的细胞,通过病毒基因拷贝数和病毒滴度检测GP5Δ84-119表达对PRRSV复制影响;通过Real-time PCR和ELISA检测病毒感染前后细胞IFN-α、IFN-β、IFN-γ的表达情况。经RT-PCR、Western blot和IFA检测,GP5Δ84-119在Marc-145细胞中获得稳定表达,将此细胞命名为Marc-145-GP5Δ84-119;细胞增殖曲线显示GP5Δ84-119的表达不影响细胞的增殖;对病毒基因拷贝数和病毒滴度检测发现GP5Δ84-119的表达可以抑制PRRSV的复制,这种抑制作用可能是通过上调IFN特别是IFN-β的表达而实现的。研究表明GP5第二胞外区在PRRSV复制中发挥重要作用。

This experiment was conducted to study the role of the second extracellular domain of glycoprotein 5,one major structural protein of porcine reproductive and respiratory syndrome virus（PRRSV）,on PRRSV replication and approach to its mechanism in Marc-145 cells.A recombinant plasmid（named as pPB-GP5Δ84-119）expressing truncated GP5 was constructed using PiggyBac Transposon System Vectors and transfected to Marc-145 cells.Marc-145 cell line stably expressing GP5Δ84-119 was obtained by puromycin resistance screening and triple subcloning and the selected cells proliferation was detected using cck-8kit.The effect of GP5Δ84-119 stable expression on PRRSV replication were determined thought detection of virus gene copy number in the infected cells and virus titers in the supernatant of infected cells.What＇s more,the mRNA and protein expression levels of IFN-α,IFN-β,IFN-γin the cells before and after PRRSV infection were also analyzed using Real-time PCR and ELISA.The results of RT-PCR,Western blot and IFA con-firmed that GP5Δ84-119 was stablely expressed in Marc-145 cells and the cells were named as Marc-145-GP5Δ84-119.The result of cell proliferation assay confirmed that the expression of GP5Δ84-119 did not affect the proliferation of Marc-145 cells.It was found that GP5Δ84-119 expression inhibited the replication of highly pathogenic PRRSV in Marc-145 cells through upregulating IFN level,especially IFN-β.These findings suggest that the second extracellular domain of GP5 plays an important role in PRRSV replication.